PT  - JOURNAL ARTICLE
AU  - Nikolaus Goessweiner-Mohr
AU  - Vadim Kotov
AU  - Matthias J. Brunner
AU  - Julia Mayr
AU  - Jiri Wald
AU  - Lucas Kuhlen
AU  - Sean Miletic
AU  - Oliver Vesper
AU  - Wolfgang Lugmayr
AU  - Samuel Wagner
AU  - Frank DiMaio
AU  - Susan Lea
AU  - Thomas C. Marlovits
TI  - Structural control for the coordinated assembly into functional pathogenic type-3 secretion systems
AID  - 10.1101/714097
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 714097
4099  - http://biorxiv.org/content/early/2019/07/24/714097.short
4100  - http://biorxiv.org/content/early/2019/07/24/714097.full
AB  - Functional injectisomes of the type-3 secretion system assemble into highly defined and stoichiometric bacterial molecular machines essential for infecting human and other eukaryotic cells. However, the mechanism that governs the regulated step-wise assembly process from the nucleation-phase, to ring-assembly, and the filamentous phase into a membrane embedded needle complex is unclear. We here report that the formation of a megadalton-sized needle complexes from Salmonella enterica serovar Typhimurium (SPI-1, Salmonella pathogenicity island-1) with proper stoichiometries is highly structurally controlled competing against the self-assembly propensity of injectisome components, leading to a highly unusual structurally-pleiotropic phenotype. The structure of the entire needle complex from pathogenic injectisomes was solved by cryo electron microscopy, focused refinements (2.5-4 Å) and co-variation analysis revealing an overall asymmetric arrangement containing cyclic, helical, and asymmetric sub-structures. The centrally located export apparatus assembles into a conical, pseudo-helical structure and provides a structural template that guides the formation of a 24-mer cyclic, surrounding ring, which then serves as a docking interface comprising three different conformations for sixteen N-terminal InvG subunits of the outer secretin ring. Unexpectedly, the secretin ring excludes the 16th protein chain at the C-terminal outer ring, resulting in a pleiotropic 16/15-mer ring and consequently to an overall 24:16/15 basal body structure. Finally, we report how the transition from the pseudo-helical export apparatus into the helical filament is structurally resolved to generate the protein secretion channel, which provides the structural basis to restrict access of unfolded effector substrates. These results highlight the diverse molecular signatures required for a highly coordinated assembly process and provide the molecular basis for understanding triggering and transport of unfolded proteins through injectisomes.